The epithelium comprising the conjunctiva is classified as a non-keratinizing stratified squamous epithelium consisting of several layers (Gipson, 1994). Goblet cells, highly specialized epithelial cells are located in the apical surface of the conjunctiva, interspersed among the layers of stratified epithelium (Wei et al.; Geggel et al.). These cells are readily identified by their extensive apical accumulation of secretory vesicles (Jeffery et al.; Huang et al.) and can occur either singly as in humans and other mammals (Kessing; Latkovic; Tseng et al.) or in clusters as found in the conjunctiva of adult rats (Srinivasan et al.). Irrespective of species, goblet cells are primarily responsible for the secretion of the inner mucous layer of the tear film, which provides a physical and chemical barrier to protect the ocular surface from dryness or other deleterious environments and/or a variety of noxious agent. (Lamberts; Nichols et al.; Gibbons; Lemp et al.) In this regard, goblet cells synthesize, store and secrete high molecular weight glycoproteins referred to as mucins, which upon secretion have the ability to hydrate and gel, producing a protective scaffolding over the ocular surface. (Chao et al.; Steuhl) Maintenance of this covering is essential to the health of the corneal and conjunctival surface. Inability or interference in the ability of goblet cells to secrete normal levels of mucin can lead to pathological abnormalities within the conjunctiva. Mucin deficiency often results as a consequence of ocular cicatricial pemphigoid, Steven Johnson syndrome, alkali burns and neurotrophic keratitis whereas overproduction of mucin due to excessive goblet cell secretion or proliferation is thought to be mediated by activated T-cells and macrophages and by a chronic conjunctivitis such as atopic keratoconjunctivitis. (Lemp, 1973; Tseng, Ophthalmol., 1984; Gilbard et al.; Lemp, 1992) These diseases and their sequellae can eventually lead to deterioration of the ocular surface.
Because the importance of the goblet cell in maintaining the integrity of the ocular surface is well recognized, a large number of structural, ultrastuctural and histochemical studies have been performed on the conjunctival epithelium in a variety of species (Latkovic; Steuhl; Setzer et al.; Moore et al.; Oduntan; Breithnach et al.). Data derived from these studies have provided valuable information regarding the development, subsequent appearance, location and function of goblet cells within the conjunctiva. In addition, they have provided information concerning the influence of environmental factors, chemical, toxin and disease upon these same goblet cell parameters.
Previous reports of systems developed to culture goblet cells in vitro are limited. Goblet cell cultures derived from airway epithelia of hamsters, rats and humans (Wu et al., 1985; Kaartinen et al.; Wu et al., 1990) have been in use for several years. By comparison, the development of systems to culture conjunctival goblet cells is still in its infancy. Among the methods which have been used to study these cells include: sectioning of conjunctival tissue combined with a battery of histochemical staining; immunocytochemical localization; transmission electron microscopy and in situ hybridization (Huang et al.; Greiner et al.; Allansmith et al.; Kinoshita et al.); histochemical staining of whole-mounted tissue (Huang et al.; Tseng et al., Ophthalmol. Vis. Sci., 1984); PAS staining of filter paper strips applied to the conjunctival surface (Adams); phalloidin labeling of excised conjunctiva (Gipson, 1997) and neutral protease removal of viable sheets of conjunctival epithelium (Geggel et al.) and growth of conjunctival cells on various substrata including natural extracellular matrix components, fibroblast feeder layers and on collagen and matrigel (Sun et al.; Rheinwald et al.; Tsai et al.). These systems are limited in that they yield indirect information. The limitations result from the information often being extrapolated from studies using whole-mounted or sectioned conjunctival tissue, or from being derived from intestinal and tracheal neoplastic cell lines, which mimic only select functions of goblet cells. Conjunctival cells have been grown from a variety of tissues including human, but no reproducible, characterized system by which goblet cells can be propagated has been reported.
Successful and consistent isolation and culture of goblet cells without altering their phenotype and/or function has been limited. In particular, normal human diploid cells have a limited proliferative lifespan in culture. In the past, it was necessary to use a variety of complex culture media as well as artificial matrices in order for the cells to attach. These techniques, however, would not always insure growth, propagation and preservation of cellular function. Therefore, there remains a need for a reliable method for culturing mammalian goblet cells.